Heterocyclic compounds and their use in the detection of nucleic acids

ABSTRACT

The invention concerns compounds of the general formula (I) in which the residues R 1  to R 7  have the meanings given in the application as well as methods for their preparation. The compounds are in particular suitable as substrates for RNA or DNA polymerases and can thus be incorporated into RNA or DNA oligonucleotides and are especially suitable for labelling and detecting nucleic acids or for DNA sequencing.

This application is a continuation of U.S. Ser. No. 10/096,786, filedMar. 11, 2002, now U.S. Pat. No. 6,875,859, which is a continuation ofU.S. Ser. No. 09/254,644, filed Jul. 2, 1999, now U.S. Pat. No.6,403,786, which is an application filed under U.S.C. 35 §371 claimingpriority to PCT/EP97/04972 filed Sep. 11, 1997, which claims priority toDE 196 37 042 filed Sep. 12, 1996, all of which are incorporated hereinby reference in their entirety for all purposes.

The invention concerns heterocyclic compounds which can be used tolabel, detect and sequence nucleic acids.

Nucleic acids are of major importance in the living world as carriersand transmitters of genetic information. Since their discovery by F.Miescher they have aroused a wide scientific interest which has led tothe elucidation of their function, structure and mechanism of action.

An important tool for explaining these connections and for solving theproblems was and is the detection of nucleic acids and namely withregard to their specific detection as well as with regard to theirsequence i.e. their primary structure.

The specific detectability of nucleic acids is based on the property ofthese molecules to interact, i.e. to hybridize, with other nucleic acidsto form base pairs by means of hydrogen bridges. Nucleic acids (probes)labelled in a suitable manner, i.e. provided with indicator groups, canthus be used to detect complementary nucleic acids (target).

The determination of the primary structure (sequence), i.e. the sequenceof the heterocyclic bases of a nucleic acid, is achieved by means ofsequencing techniques. Knowledge of the sequence is in turn a basicrequirement for a targeted and specific use of nucleic acids formolecular biological problems and working techniques.

The sequencing also ultimately utilizes the principle of specifichybridization of nucleic acids to one another. As mentioned abovelabelled nucleic acid fragments are also used for this.

Hence a suitable labelling of nucleic acids is an essential prerequisitefor any detection method.

At an early period radioactive labelling was mainly used with suitableisotopes such as ³²P or ³⁵S. However, the disadvantages of usingradioactive reagents are obvious: such work requires special roomfacilities and licences as well as a controlled and elaborate disposalof the radioactive waste. The reagents for radioactive labelling areexpensive. It is not possible to store such labelled probes for longperiods due to the short half-life of the above-mentioned nuclides.

Therefore many attempts have been made in recent years to circumventthese serious disadvantages i.e. to get away from using a radioactivelabel. However, the high sensitivity of this type of label should beretained as far as possible.

Major advances have in fact already been achieved [see e.g.Nonradioactive Labeling and Detection of Biomolecules (Kessler, C.,publ.) Springer Verlag Berlin, Heidelberg 1992].

An essential requirement for any detection of a nucleic acid is theprior labelling. As indicated above it is desirable to achieve this in anon-radioactive manner. Whereas radioactive labelling of nucleic acidsis usually carried out by the enzymatically catalysed incorporation ofappropriate radioactive nucleoside triphosphates, non-radioactivelabelling has to be achieved by incorporating a suitable signal orreporter group.

Haptens (such as biotin or digoxigenin), enzymes (such as alkalinephosphatase or peroxidase) or fluorescent dyes (such as fluorescein orrhodamine) have, among others, mainly proven to be suitable asnon-radioactive indicator molecules. These signal groups can be attachedto or incorporated in nucleic acids by various methods.

A relatively simple procedure is for example to label the 5′ end of anoligonucleotide provided with a terminal amino group by means ofactivated indicator molecules of the above-mentioned type. However, thisonly allows the introduction of one or a few indicator molecules intoonly a low molecular oligomer whereas a denser labelling of longerchain, high molecular nucleic acids with the aim of achieving a highsensitivity usually has to be accomplished by incorporating nucleosidetriphosphates provided with reporter groups by means of polymerases asin a de novo synthesis.

Such current methods are known to a person skilled in the art as nicktranslation [Rigby, P. W. et al., (1977), J. Mol. Biol. 113, 237] andrandom primed labeling [Feinberg, A. P. & Vogelstein, B. (1984) Anal.Biochem. 137, 266]. A further method is the so-called 3′-tailingreaction with the aid of the enzyme terminal transferase. [e.g. Schmitz,G. et al (1991) Anal. Biochem. 192, 222].

The nucleoside triphosphates which have been previously used in thesemethods are almost exclusively appropriately modified derivatives of theheterocyclic bases adenine, guanine, cytosine and thymine in thedeoxyribonucleotide series or adenine, guanine, cytosine and uracil inthe ribonucleotide series. Such derivatives are described for example byLanger et al. in Proc. Natl. Acad. Sci. USA 78, 6635 (1981), Mühleggeret al. Biol. Chem. Hoppe-Seyler 371, 953 (1990) and in EP 0 063 879. Inthis case the building blocks which occur naturally in DNA and RNA areused in a labelled form i.e. provided with signal groups.

The main disadvantages of these N-nucleosides is that the N-glycosidicbond is sensitive to acidic pH conditions and they can be degraded bynucleases.

Furthermore individual C-nucleosides (see e.g. Suhadolnik, R. J. in“Nucleoside Antibiotics”., Wiley-Interscience, New York 1970) and theiruse in the therapeutic (antiviral or cancerostatic) field has also beenknown for a long time. In addition fluorescent C-nucleoside derivativesand their incorporation into DNA and RNA oligonucleotides has beendescribed (WO 93/16094). The so-called intrinsic fluorescence of thesenucleosides is, however, many times lower with regard to quantum yieldthan that of the special fluorophores such as fluorescein orcorresponding rhodamine derivatives. A further disadvantage of theself-fluorescent C-nucleosides is their comparatively low excitation andemission wavelengths. As a result detection systems which are based onsuch derivatives only have a low sensitivity of detection and on theother hand influences of the measuring environment which interferespectrally (such as biological material, autofluorescence of gelmatrices etc.) have a very major effect. Hence the known nucleosides andnucleoside derivatives have a series of disadvantages which especiallyhave an adverse effect on the detection of nucleic acids. Hence theobject of the invention is to provide nucleoside derivatives modifiedwith signal groups for the detection of nucleic acids which do not havethe afore-mentioned disadvantages i.e. in particular are more stable andat the same time capable of being processed enzymatically and aresuitable for the detection of nucleic acids at a practicable wavelength.

The object is achieved by heterocyclic compounds of the general formulaI

in which

-   R₁ and R₂ can be the same or different and represent hydrogen,    oxygen, halogen, hydroxy, thio or substituted thio, amino or    substituted amino, carboxy, lower alkyl, lower alkenyl, lower    alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a    reporter group, R₃ and R₄ each represent hydrogen, hydroxy, thio or    substituted thio, amino or substituted amino, lower alkyloxy, lower    alkenoxy, lower alkinoxy, a protecting group or a reporter group,-   R₅ represents hydrogen, hydroxy, thio or substituted thio, amino or    a substituted amino group, a reactive trivalent or pentavalent    phosphorus group such as e.g. a phosphoramidite or H-phosphonate    group, an ester or amide residue that can be cleaved in a suitable    manner or a reporter group,-   R₄ and R₅ together form a further bond between C-2′ and C-3′ or an    acetal group,-   R₆ represents hydrogen or a hydroxy, thio or substituted thio, amino    or substituted amino group,-   R₇ represents hydrogen, a monophosphate, diphosphate or triphosphate    group or the alpha, beta or gamma thiophosphate analogue of this    phosphoric acid ester or a protective group    as well as possible tautomers and salts thereof.

X denotes methylene or methine substituted with halogen, hydroxy, thioor substituted thio, amino or substituted amino, carboxy, lower alkyl,lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl,aralkyloxy or a reporter group, or oxygen and n=0 or 1, Z denotesnitrogen or carbon provided that if Z denotes nitrogen, m is zero (0)and if X represents methylene, substituted methylene or substitutedmethine, Z cannot be carbon and if X denotes oxygen, Z cannot benitrogen.

All detectable groups come into consideration as a reporter group suchas in particular haptens, a fluorophore, a metal-chelating group, alumiphore, a protein or an intercalator.

Those compounds of the general formula I are preferred in which theacetal group of the residues R₄ and R₅ is substituted with a reportergroup. The reporter group can be bound directly or indirectly i.e. via alinker group.

In addition those compounds of the general formula I have proven to beparticularly suitable in which R₁ can represent oxygen, R₂ can representhydrogen or a reporter group, R₃ and R₄ can represent hydrogen, R₅ canrepresent hydroxy, hydrogen, a reactive trivalent or pentavalentphosphorus group, R₆ can represent hydrogen and R₇ can representhydrogen, monophosphate, diphosphate or triphosphate groups.

Compounds of the general formula I are also preferred in which thereporter group is bound to the heterocyclic or tetrahydrofuran ring bymeans of a so-called linker group. Suitable linker groups are known to aperson skilled in the art (see e.g. Mühlegger, K. et al. (1990) Biol.Chem. Hoppe-Seyler 371, 953-965 or Livak, K. J. et al. (1992) Nucl.Acids Res. 20, 4831-4837).

Compounds of the general formula I are additionally preferred in whichR₁ represents hydrogen, hydroxy, an amino group, an optionallysubstituted amino group or a reporter group, R₂ represents an optionallysubstituted amino group or a reporter group, R₃ represents hydrogen, R₄represents hydrogen, hydroxy, amino or substituted amino, loweralkyloxy, lower alkenoxy, lower alkinoxy, R₅ represents hydrogen,hydroxy, thio, an optionally substituted amino group, a phosphoramiditeor a reporter group, R₄ and R₅ together represent an acetal group, R₆represents hydrogen and R₇ represents a triphosphate group.

Compounds of formula I are also preferred in which X denotes oxygen andat the same time Z represents carbon substituted with R₂ or Z denotesnitrogen and at the same time X represents methylene or methinesubstituted with amino or substituted amino, carboxy or with a reportergroup.

A further preferred embodiment is compounds according to formula I inwhich X=0 and Z represents methine substituted with amino or substitutedamino, carboxy or with a reporter group.

The compounds according to the invention can be synthesized in variousways. In some cases one can start with naturally occurring precursorssuch as for example3-(3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-pyrrol-2,5-dioneor3-(3,4-dihydroxy-5-hydroxy-methyl-tetrahydrofuran-2-yl)-oxazine-2,6-dione.The important 3-(3-deoxy-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)derivatives are synthesized from these precursors by deoxygenationpreferably according to Barton (Barton, D. H. R & Motherwell, W. B.(1981) Pure Appl. Chem. 53, 15).

In addition the chemical synthesis of the new heterocyclic compounds canfor example be carried out as for example described in detail by K. A.Watanabe in “Chemistry of Nucleosides and Nucleotides” 3, 421-535 (L. B.Townsend, publ.) Plenum Press, New York and London, 1994.

Other syntheses of the said starting compounds have for example beendescribed by Hosmane, R. S. et al. in Bioorg. & Med. Chem. Lett. 3, 2847(1993) and by Townsend, L. B. et al. in Tetrahedron Lett. 36, 8363(1995).

The use of the compounds according to the invention to label nucleicacids with diverse, defined signal groups and hence to detect andsequence nucleic acids has proven to be particularly advantageous.

The substances according to the invention of the general formula I havea number of advantages especially compared to the classical nucleosidesand nucleotides such as adenosine, guanosine, cytidine, thymidine,uridine etc. and their corresponding phosphoric acid esters.

One advantage is chemical stability i.e. towards acidic pH conditions. Afurther major advantage is the stability of these compounds towardsenzymatic degradation by endonucleases and exonucleases. These enzymesare present in biological material and can severely interfere with thenucleic acid detection. On the other hand it is known that DNA and RNApolymerases are critical with regard to the acceptance of more or lessmodified nucleoside 5′-triphosphates i.e. with regard to the recognitionand incorporation of such nucleotides as substrates in de novosynthesis. Experience has shown that the attachment of signal groups tonucleotides influences in particular their incorporation andincorporation rate.

The fact that the derivatives according to the invention can beincorporated by suitable polymerase into nucleic acids in a veryefficient manner such as e.g. by the aforementioned methods of nicktranslation or of random primed labelling cannot be inferred from theprior art and must therefore be regarded as surprising for a personskilled in the art.

The said methods are used quite generally in nucleic acid detection e.g.for quantitative detection using blotting techniques on membranes oralso in microtitre plates.

In sequencing, i.e. detecting the sequence of a nucleic acid, acomplementary opposite strand is newly synthesized on the nucleic acidto be sequenced with the aid of a short (start) oligonucleotide (primer)and the addition of labelled nucleoside triphosphates and a polymerase,subsequently so-called termination reactions are carried out and thenucleic acid fragments that are generated in this process are separatedby gel chromatography.

In principle the same occurs in the cell in the in situ hybridization todetect certain genes or genome sections i.e. the specific incorporationof labelled nucleotides.

The above-mentioned primers i.e. short-chain oligonucleotides shouldform stable base pairs with the template strand as well as not beattacked by endogenous nucleases in order to ensure an optimal function.

This is fulfilled by oligonucleotides which contain the compoundsaccording to the invention as building blocks instead of the classicalnucleosides.

The same applies to longer chain polynucleotides and nucleic acids whichcontain such building blocks. These are also a subject matter of thepresent invention.

Corresponding oligonucleotides and their preparative precursors in theform of so-called phosphoramidites and H-phosphonates are therefore alsoa subject matter of the invention.

Oligonucleotides are nowadays usually produced by known methods inautomated DNA/RNA synthesizers by solid phase synthesis.

Such methods of synthesis are based essentially on the stepwise reactionof the aforementioned phosphoramidites or H-phosphonates and hence thecontinuous linkage of these monomeric building blocks to form oligomers(see e.g. T. Brown & D. J. S. Brown in Oligonucleotides and Analogues-APractical Approach, (1991) (Eckstein, F., publ. IRL Press at OxfordUniversity Press, Oxford, N.Y., Tokyo).

Legend

FIG. 1:

I and II denote pBR 328-DNA labelled by DIG-dUTP incorporation(standard) and III denotes pBR 328-DNA labelled by the compound3-(4-hydroxy-5triphosphoryl-tetrahydrofuran-2-yl)-4-(digoxigeninyl-3-O-succiny-aminocaproylamino-pentyl)-amino-pyrrol-2,5-dione synthesized accordingto example 6. They are applied to the gel at concentrations of 10 to0.01 pg.

The invention is further elucidated by the following examples:

EXAMPLE 13-(4-Hydroxy-5-hydroxy-methyl-tetrahydrofuran-2-yl)-pyrrole-2,5-dione

The compound was prepared in a de novo synthesis according to Hosmane,R. S. et al. Bioorg. & Med. Chem. Lett. 3, 2847 (1993).

Alternatively it can be obtained by Barton deoxygenation [Barton, D. H.R. & Motherwell, W. B. (1981) Pure Appl. Chem. 53, 15] from the3,4-dihydroxy derivative (showdomycin) obtained by fermentation.

EXAMPLE 23-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-bromo-pyrrole-2,5-dione

213 mg (1 mmol)3-(4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-pyrrol-2,5-dioneobtained according to example 1 is dissolved in 25 ml water saturated atRT with bromine and stirred for 3 hours at room temperature. Afterwardsonly a small amount of starting material is observed in the TLC. Thesolution is freed of excess bromine in a vacuum, adjusted to pH 7 andevaporated to an oil. It is taken up in a small amount of methanol andseparated on a silica gel column with a mixture of chloroform/methanol8:2. After evaporating the fractions, 140 mg (48%) of a pale yellow oilis obtained.

Elemental analysis: for C₉H₁₀NO₅Br (MW292.2): C_(calc)36.9; H_(calc)3.4;N_(calc)4.8; Br_(calc)27.4; C_(found)37.35; H_(found)3.6; N_(found)4.5;Br_(found)27.8.

EXAMPLE 33-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-(1,5-diaminopentyl)-pyrrole-2,5-dione

140 mg (ca. 0.5 mmol) of the bromine compound from example 2 isdissolved in 50 ml ethanol, admixed with 1.75 g (ca 10 mmol)diaminopentane dihydrochloride and heated to reflux for 5 hours.Afterwards the conversion is almost quantitative (compared to the lowerspot of the bromine compound, ninhydrin positive) according to TLC(silica gel, chloroform/methanol 8:2). The reaction mixture isevaporated in a vacuum and used in example 4 without furtherpurification.

EXAMPLE 43-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-(N-trifluoroacetamidopentyl)-amino-pyrrole-2,5-dione

The oily residue from example 3 (ca. 2 g) is dissolved in 50 mlanhydrous pyridine, undissolved material is removed by suctionfiltration and the filtrate is evaporated to dryness in a vacuum. It istaken up in 50 ml absolute pyridine and 0.75 ml (ca. 5 mmol)trifluoroacetic anhydride is added. After standing for 5 hours at RT theacylation is complete according to TLC. Subsequently the reactionsolution is evaporated in a vacuum and coevaporated three times withmethanol. It is taken up in ca. 20 ml ethanol, filtered andchromatographed on silica gel with a mixture of chloroform/methanol(9:1). The combined fractions are evaporated, the residue is taken up indioxane and lyophilized. 110 mg (53% of theory) of the desired compoundis obtained.

Elemental analysis for C₁₆H₂₃N₃O₆F₃ (MW 410.4): C_(calc)46.8;H_(calc)5.6; N_(calc)10.2; F_(calcl)13.9; C_(found)47.35; H_(found)5.9;N_(found)10.5; F_(found)13.8.

EXAMPLE 5 3-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-4-(N-trifluoroacetamidopentyl)-amino-pyrrole-2,5-dione

40 mg (0.1 mmol) of the protected nucleoside from example 4 is convertedby phosphorylation with POCl₃ into the 5′-monophosphate according to themethod of Yoshikawa et al. [Tetrahedron Lett. 50, 5065 (1967)]; thedesired triphosphate is obtained from this in a yield of 30 mg (46%)according to the method of Hoard & Ott [J. Am. Chem. Soc. 87, (1965)]after activation with carbonyldiimidazole and reaction withpyrophosphoric acid and subsequent ion exchange chromatography on DEAESephadex.

³¹P-NMR (0.1 M EDTA/D₂O/Eth₃N): −5.2 (d,P-γ); −10.3 (d, P-α); −21.0(t,P-β).

EXAMPLE 63-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-4-(N-fluoresceinyl-carboxamido-pentyl)-amino-pyrrole-2,5-dione

25 mg (0.038 mmol) of the trifluoracetyl-protected compound from example5 is allowed to stand for 1 h at RT in 5 ml concentrated ammoniasolution and subsequently evaporated in a vacuum. The residue is takenup in 5 ml 0.1 M borate buffer, pH 8.5 and admixed with a solution of 25mg (0.05 mmol) 5(6)-carboxy -fluorescein-N-hydroxy-succinimide ester in5 ml amine-free dimethyl formamide. It is allowed to stand overnight atroom temperature. The reaction mixture is applied to a DEAE Sephadexcolumn (30×1 cm) and eluted with a linear LiCl gradient (200 ml H₂O to0.4 M LiCl). After combining the appropriate fractions, evaporating,precipitating the concentrate in acetone/ethanol (2:1) and drying, 25 mg(ca. 50%) of the title substance is obtained.

Spectral data (0.1 M phosphate buffer, pH 9.0:

-   -   excitation_(max) [nm]: 495;    -   emission_(max): [nm]: 521

The3-(4-hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-4-(digoxigeninyl-3-O-succinyl-aminocaproylamino-pentyl)-amino-pyrrole-2,5-dionewas prepared in a corresponding manner by reacting the compound fromexample 5 with digoxigenin-3-O-succinyl-aminocaproicacid-N-hydroxy-succinimide ester.

EXAMPLE 73-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1,3-oxazine-2,6-dione

The compound was obtained by Barton deoxygenation [Barton, D. H. R. &Motherwell, W. B. (1981) Pure Appl. Chem. 53, 15] from the 3,4-dihydroxyderivative (oxazinomycin) obtained by fermentation.

EXAMPLE 83-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-bromo-1,3-oxazine-2,6-dione

The derivative was obtained by brominating the starting compound fromexample 7 as described in example 2.

EXAMPLE 93-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-(1,5-diaminopentyl)-1,3-oxazine-2,6-dione

150 mg (0.5 mmol) of the bromine compound from example 8 was convertedinto the title compound according to the method of example 3. This wasfinally reacted with fluorescein-labelled triphosphate without furtherpurification according to the methods of examples 4, 5 and 6.

EXAMPLE 103-(4-Hydoxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-2,6-diamino-5-chloro-pyrazine

The derivative was synthesized according to Townsend, L. B. et al.Tetrahedron Lett. 36, 8363 (1995).

EXAMPLE 113-(4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-2,6-diamino-5-chloro-pyrazine

264 mg (1 mmol) 3-(4-Hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-chloro-pyrazine from example 10was subjected to a deamination reaction in a mixture of 50 ml 80% aceticacid and 700 mg (10 mmol) NaNO₂. After standing for 5 hours at RT, thereaction was almost complete according to TLC. 2 g urea was added to thereaction mixture to destroy excess nitrite and stirred for a furtherthree hours at RT. Afterwards the solution was applied to an activatedcarbon column (Carboraffin C, ca. 50 ml volume), adequately washed andthe desired product was eluted with ethanol/water/ammonium. 230 mg (ca.87%) of a viscous oil resulted after evaporation which was used in thenext stage without further purification.

EXAMPLE 123-(4-Hydroxy-5-hydroxy-methyl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-(1,8-diamino-3,6-dioxa-octyl)-pyrazine

200 mg (0.75 mmol) of the oil from example 11 was dissolved in 30 mlethanol, 555 mg (3.75 mmol) 1,8-diamino-3,6-dioxa-octane was added andit was heated for 3 hours to ca. 60° C.

Subsequently the solvent and amine were removed in an oil pump vacuumand the residue was further reacted without further purification byreaction with trifluoroacetic anhydride in pyridine as described inexample 4.

EXAMPLE 133-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-[N-trifluoro-acetamido-(3,6-dioxa)-octyl]-amino-pyrazine

150 mg of the trifluoracetylated derivative from example 12 wasconverted into the title compound according to Yoshikawa and Hoard & Ottas described in example 5. The desired triphosphate was obtained in ayield of 120 mg (40%) after ion exchange chromatography on DEAESephadex.

³¹P-NMR (0.1 M EDTA/D₂O/Eth₃N): −5.1 (d,P-γ); −10.6 (d,P-α); −20.8(t,P-β).

EXAMPLE 143-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-[N-tetramethyl-rhodaminyl-5,6-carboxamido-(3,6-dioxa)-octyl]-amino-pyrazine

20 mg of the triphosphate from example 13 were reacted—after cleavage ofthe trifluoroacetyl protective group with ammonia solution (as describedin example 6)—with 20 mg tetramethylrhodamine-5(6)-carboxylic acid-N-hydroxy-succinimide ester in 0.1 M sodium borate buffer, pH 8.5/DMF asdescribed in example 6 and purified. 12 mg of the TMR-labelled productwas obtained.

Spectral data (0.1 M Na-borate buffer, pH 8.5):

-   -   excitation_(max) [nm]: 551;    -   emission_(max): [nm]: 575

EXAMPLE 15

Non-radioactive DNA labelling and detection by incorporation of3-(4-hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-4-(digoxigeninyl-3-O-succinyl-aminocaproylamino-pentyl)-amino-pyrrole-2,5-dione The DNA labelling andthe DNA detection were carried out using the commercially available kitfrom the Boehringer Mannheim Company (order No. 1093 657). All essentialprocess steps are described in the working instructions.

For the labelling reaction the DIG-dUTP in the dNTP mixture in the kitwas substituted by a3-(4-hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-4-(digoxigeninyl-3-O-succinyl-aminocaproylamino-pentyl)-amino-pyrrole-2,5-dione(synthesized as described in example 6).

The immunological detection reaction showed that incorporation of theinventive compound of example 6 resulted in a detection sensitivity ofthe labelled DNA which is similar to the use of DIG-dUTP.

The result which demonstrates the detection and the achieved sensitivityof the system is shown in FIG. 1.

1. A nucleotide analog of the formula

in which R₁ is hydrogen, halogen, hydroxy, thio, carboxy, lower alkyl,lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl,aralkyloxy or a reporter group, R₃ and R₄ each is independentlyhydrogen, hydroxy, thio, amino, lower alkyloxy, lower alkenoxy, loweralkinoxy, an ester, amide or a reporter group, R₅ is hydrogen, hydroxy,thio, amino, a trivalent phosphorus group, a pentavalent phosphorusgroup, an ester, amide or a reporter group, or R₄ and R₅ together form afurther bond between C-2′ and C-3′ or an acetal group, R₆ is hydrogen,hydroxy, thio, or amino, R₇ is hydrogen, a monophosphate, a diphosphatea triphosphate group, an alpha, beta or gamma thiophosphate analogue ofa phosphate group, an ester or an amide group, X is methine substitutedwith halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl,lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or areporter group, each reporter group is independently selected from thegroup consisting of a hapten and a fluorophore, optionally linked via alinker group; and salts thereof.
 2. The nucleotide analog of claim 1, inwhich R₁ is hydroxy, R₃ and R₄ are hydrogen, R₅ is hydroxy, hydrogen, atrivalent phosphorus group, or a pentavalent phosphorus group, R₆ ishydrogen and R₇ is hydrogen, a mono-phosphate group, di-phosphate group,or a triphosphate group.
 3. The nucleotide analog of claim 1, wherein R₁is hydrogen, hydroxy, or a reporter group, R₃ is hydrogen, R₄ ishydrogen, hydroxy, amino, lower alkyloxy, lower alkenoxy, or loweralkynoxy, R₅ is hydrogen, hydroxy, thio, a phosphoramidite or a reportergroup, or R₄ and R₅ together are an acetal group, R₆ is hydrogen and R₇is a triphosphate group.
 4. The nucleotide analog of claim 1, in which Xis methine substituted with amino, carboxy or a reporter group.
 5. Thenucleotide analog of claim 1, wherein the acetal group of the residuesR₄ and R₅ is substituted with a reporter group.
 6. The nucleotide analogof claim 1, in which the reporter group is linked to said compound via alinker group.
 7. An isolated oligonucleotide comprising at least onenucleotide analog of the formula:

wherein R₁ is hydrogen, oxygen, halogen, hydroxy, thio, carboxy, loweralkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy,aralkyl, aralkyloxy or a reporter group, R₂ is oxygen, halogen, hydroxy,thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl,lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, R₃ andR₄ each independently is hydrogen, hydroxy, thio, amino, lower alkyloxy,lower alkenoxy, lower alkinoxy, an ester, an amide group or a reportergroup, R₅ is hydrogen, hydroxy, thio, amino, a trivalent phosphorusgroup, a pentavalent phosphorus group, an ester, an amide or a reportergroup, R₄ and R₅ together form a further bond between C-2′ and C-3′ oran acetal group, R₆ is hydrogen, a hydroxy, thio, or amino group, R₇ ishydrogen, a monophosphate, a diphosphate a triphosphate group an alpha,beta or gamma thiophosphate analogue of a phosphate group, an ester oran amide group; X is methylene or methine substituted with halogen,hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, loweralkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or areporter group, each reporter group is independently selected from thegroup consisting of a hapten and a fluorophore; Z is nitrogen or carbon,provided that if Z denotes nitrogen, m is 0, and if X is methylene,substituted methylene or substituted methine, Z is nitrogen and at leastone of R₅ and R₇ indicates the point of attachment to theoligonucleotide.
 8. A isolated nucleic acid comprising at least onenucleotide analog of the formula

wherein R₁ is hydrogen, oxygen, halogen, hydroxy, thio, carboxy, loweralkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy,aralkyl, aralkyloxy or a reporter group, R₂ is oxygen, halogen, hydroxy,thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl,lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, R₃ andR₄ each independently is hydrogen, hydroxy, thio, amino, lower alkyloxy,lower alkenoxy, lower alkinoxy, an ester, an amide group or a reportergroup, R₅ is hydrogen, hydroxy, thio, amino, a trivalent phosphorusgroup, a pentavalent phosphorus group, an ester, an amide or a reportergroup, R₄ and R₅ together form a further bond between C-2′ and C-3′ oran acetal group, R₆ is hydrogen, a hydroxy, thio, or amino group, R₇ ishydrogen, a monophosphate, a diphosphate a triphosphate group an alpha,beta or gamma thiophosphate analogue of a phosphategroup, an ester or anamide group; X is methylene or methine substituted with halogen,hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, loweralkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or areporter group, each reporter group is independently selected from thegroup consisting of a hapten and a fluorophore; Z is nitrogen or carbon,provided that if Z denotes nitrogen, m is 0, and if X is methylene,substituted methylene or substituted methine, Z is nitrogen and at leastone of R₅ and R₇ indicates the point of attachment to theoligonucleotide.
 9. A reaction mixture comprising: a) a nucleotideanalog of the general formula

in which: R₁ and R₂ are the same or different and represent hydrogen,oxygen, halogen, hydroxy, thio, amino, carboxy, lower alkyl, loweralkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl,aralkyloxy or a reporter group, R₃ and R₄ each represent hydrogen,hydroxy, thio, amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, aprotecting group or a reporter group, R₅ represents hydrogen, hydroxy,thio, amino, a reactive trivalent or pentavalent phosphorus group, anester or amide residue that can be cleaved or a reporter group, or R₄and R₅ together form a further bond between C-2′ and C-3′ or an acetalgroup, R₆ represents hydrogen or a hydroxy, thio, or amino group, R₇represents hydrogen, a monophosphate, diphosphate or triphosphate groupor the alpha, beta or gamma thiophosphate analogue of said phosphategroup or a protective group, and tautomers and salts thereof, Xrepresents methine substituted with halogen, hydroxy, thio, amino,carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, loweralkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, eachreporter group is independently selected from the group consisting of ahapten and a fluorophore; and Z represents nitrogen or carbon, providedthat if Z represents nitrogen, m is zero (0) Z is nitrogen; and b) apolymerase.
 10. The reaction mixture of claim 9, further comprising anoligonucleotide primer.
 11. The reaction mixture of claim 10, whereinthe oligonucleotide primer comprises the nucleotide analog wherein R₁ ishydrogen, oxygen, halogen, hydroxy, thio, amino, carboxy, lower alkyl,lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl,aralkyloxy or a reporter group, R₂ is oxygen, halogen, hydroxy, thio,amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, loweralkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, R₃ and R₄each independently is hydrogen, hydroxy, thio, amino, lower alkyloxy,lower alkenoxy, lower alkinoxy, an ester, an amide group or a reportergroup, R₅ is hydrogen, hydroxy, thio, amino, a trivalent phosphorusgroup, a pentavalent phosphorus group, an ester, an amide or a reportergroup, R₄ and R₅ together form a further bond between C-2′ and C-3′ oran acetal group, R₆ is hydrogen, a hydroxy, thio, or amino group, R₇ ishydrogen, a monophosphate, a diphosphate a triphosphate group an alpha,beta or gamma thiophosphate analogue of a phosphate group, an ester oran amide group; X is methine substituted with halogen, hydroxy, thio,amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, loweralkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, eachreporter group is independently selected from the group consisting of ahapten and a fluorophore; Z is nitrogen or carbon, provided that if Zdenotes nitrogen, m is 0 and at least one of R₅ and R₇ indicates thepoint of attachment to the oligonucleotide.
 12. The reaction mixture ofclaim 9, further comprising a target nucleic acid sequence.
 13. Thereaction mixture of claim 12, further comprising an oligonucleotideprimer.
 14. The reaction mixture of claim 13, wherein theoligonucleotide primer comprises the nucleotide analog wherein R₁ ishydrogen, oxygen, halogen, hydroxy, thio, amino, carboxy, lower alkyl,lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl,aralkyloxy or a reporter group, R₂ is oxygen, halogen, hydroxy, thio,amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, loweralkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, R₃ and R₄each independently is hydrogen, hydroxy, thio, amino, lower alkyloxy,lower alkenoxy, lower alkinoxy, an ester, an amide group or a reportergroup, R₅ is hydrogen, hydroxy, thio, amino, a trivalent phosphorusgroup, a pentavalent phosphorus group, an ester, an amide or a reportergroup, R₄ and R₅ together form a further bond between C-2′ and C-3′ oran acetal group, R₆ is hydrogen, a hydroxy, thio, or amino group, R₇ ishydrogen, a monophosphate, a diphosphate a triphosphate group an alpha,beta or gamma thiophosphate analogue of a phosphategroup, an ester or anamide group; X is methine substituted with halogen, hydroxy, thio,amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, loweralkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, eachreporter group is independently selected from the group consisting of ahapten and a fluorophore; Z is nitrogen or carbon, provided that if Zdenotes nitrogen, m is 0 and at least one of R₅ and R₇ indicates thepoint of attachment to the oligonucleotide.
 15. The nucleotide analog ofclaim 1, wherein R₁ is hydroxy.
 16. A nucleotide analog selected fromthe group

consisting of:3-(4-Hydroxy-5-hydroxy-methyl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-(1,8-diamino-3,6-dioxa-octyl)-pyrazine;

3-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-[N-trifluoro-acetamido-(3,6-dioxa)-octyl]-amino-pyrazine;and

3-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-[N-tetramethyl-rhodaminyl-5,6-carboxamido-(3,6-dioxa)-octyl]-amino-pyrazine.17. The nucleotide analog of claim 1, wherein the hapten is selectedfrom the group consisting of: biotin and digoxigenin.
 18. The nucleotideanalog of claim 1, wherein the fluorophore is selected from the groupconsisting of: fluorescein and rhodamine.